Buccal formulation of avanafil with enhanced bioavailability and prolonged duration

ABSTRACT

A buccal tablet formulation has a polyvinylpyrrolidone K-90 as a solid dispersion polymer, a hydroxypropyl methylcellulose as a mucoadhesive polymer, a sodium deoxycholate as mucopenetration enhancer, a porous silicon dioxide (e.g., FujiSil), mannitol, and avanafil. The ratio of PVP K-90 to AVA is approximately 2:1 (e.g., 2.3:1 to 1.7:1) in the tablet. Methods of making the buccal tablet with enhanced bioavailability and prolonged duration and methods of using the formulation for the treatment of erectile dysfunction are also provided.

FIELD OF INVENTION

The invention is generally related to the formulation of avanafil as abuccal tablet with enhanced bioavailability and a prolonged duration ofaction. Specifically, this formulation embodies a buccal route ofadministration to avoid hepatic first-pass metabolism and improve thedrug bioavailability. This approach helps minimize the undesirable sideeffect in patients with erectile dysfunction.

BACKGROUND OF THE INVENTION

Impotence/erectile dysfunction (ED) is one of the most widely recognizeddiseases in male sexual dysfunction, with studies showing that 1/3 ofAmerican men over age 40 are affected with this disease (1,2). In Arabcountries, recent studies showed that the prevalence of ED is more than40% and is linked to various risk factors such as age, obesity, lack ofactivity, smoking, and diabetes mellitus complications (3,4). Locally inJeddah, a cross-sectional, multi-clinical study showed that more than70% of the participants in the study showed moderate to severe EDsymptoms (5). It is not commonly seen as a life-threatening illness, butit is meticulously linked to several necessary physical conditions andcan impact psychosocial well-being. Therefore, ED affects patients'quality of life (6). The main goals of ED management are to monitor andminimize risk factors associated with organic cardiovascular and toregain the ability to get an efficient penile erection and sustain it.In order to effectively treat the condition, the etiology must beidentified and treated rather than just the symptoms. (7).Phosphodiesterase type 5 inhibitors (PDE5-Is) are orally active andself-administered drugs for the treatment of ED that are used as neededbefore sexual intercourse (8). Avanafil (AVA) is a second-generation andhighly selective PDE5-I commonly used in the treatment of ED. In 2012,it was approved by the United States Food and Drug Administration(USFDA), and in the following year, it was approved by the EuropeanMedicines Agency (EMA) (9). AVA is subject to significant first-passmetabolism through the human cytochrome P450 enzyme system (8).According to the Biopharmaceutical Classification System (BCS), it isclassified as a Class II drug; thus, its dissolution is therate-limiting step for its absorption, which leads to low oralbioavailability. Also, AVA absorption is altered in the presence of foodwhich delays the time required to reach its maximum serum (9). As aresult, modifying AVA's solubility, as well as selecting a differentdelivery method, may have an impact on bioavailability.

Buccal administration has attracted great attention due to its rapidonset of action and high bioavailability compared to the oral route.This route avoids first-pass metabolism and gastrointestinal tract (GIT)degradation so that it may alleviate possible side effects of the drug(10). The highly vascularized oral mucosa offers a substantialopportunity for the drugs to be absorbed, as they can gain direct accessvia capillaries and venous drainage to the systemic circulation,circumventing hepatic metabolism. Also, the oral cavity suggests aconstant and friendly physiological environment for drug delivery thatis maintained by continuous salivary secretion. Moreover, the fluidityof the saliva, low mucin content, absence of proteases, and limitedenzymatic activity represent additional advantages for the systemicdelivery of drugs after buccal administration (11). Finally, the higheroverall permeability of the buccal and sublingual mucosa compared toother mucosae of the mouth is another advantageous characteristic fordrug delivery. The oral mucosa is 4-4000 times more permeable than theskin, as water permeation is ten times higher than the skin (12).

Scientific research continues to improve drug delivery across the oralmucosa. Numerous conventional and novel drug delivery systems have beendeveloped for buccal drug delivery. These include; sprays, liquids(solutions or suspensions), semisolids (hydrogels), and solids such astablets/lozenges (including lyophilized and bio-adhesive) (13), chewinggums, and patches/films. The advances in buccal delivery systems focuson achieving the therapeutic effect by this route, overcoming thechallenging environment in the oral cavity, and finding a compromisebetween patient compliance and clinical benefits. Buccal deliverysystems can be designed as a fast, delayed, or controlled delivery ofdrugs for either local applications or systemic drug delivery (14). Thelatter is usually aimed at delivering the drug in one of three mainways: rapid release, pulsatile release (with rapid onset followed by aphase where the drug levels are maintained within the therapeuticwindow), or modified release for an extended period, depending on theaddition of different excipients like permeation enhancers or releaseretardants in the formulations. Sodium deoxycholate is an excellentexample of a permeation enhancer that has been shown to increasepermeability and, subsequently, systemic delivery, as previouslymentioned (15,16).

Additionally, the buccal dosage form's mucoadhesive properties shouldgain much concern to resist the swallowing action and control the drugrelease from the buccal mucosa. Mucoadhesive properties can beaccomplished using anionic, cationic, and nonionic polymers.Furthermore, stronger mucoadhesive properties have been achieved byusing both anionic and cationic polymers, but using anionic polymers canbe advantageous due to their lower toxicity (17). Typical examples ofanionic mucoadhesive polymers include polyacrylic acid, carbomers,polycarbophil, sodium carboxymethyl cellulose, alginate, and pectin(15). On the other hand, chitosan and hydroxypropyl methylcelluloserepresent the most widely used cationic and nonionic polymers,respectively (15,18,19).

To sum up, it is of great importance to develop novel strategies thatdeliver therapeutic substances, through a convenient and/or advanceddelivery system, with improved efficacy and bioavailability, reduceddosage frequency and undesirable side effects, improved patientcompliance, and commercialization potential.

SUMMARY OF THE INVENTION

As a result, we are investigating a novel approach to develop an AVAdosage form capable of avoiding the hepatic first-pass effect andachieving high therapeutic efficacy. Improving AVA solubility will helpaccomplish these goals. Therefore, this study aims to develop anoptimized buccal tablet formulation loaded with physically modified AVAform, utilizing the suitable carrier, to enhance the drug solubility,bypass hepatic metabolism, and improve the drug bioavailability andefficacy as well as prolong the duration of action. It is worthmentioning that our AVA buccal formulation is the first dosage form forAVA to be administered via the buccal route till the present time. Onlyfive patents (20-24) and nine research articles (5,25-32) revealeddifferent AVA formulations/dosage forms for pharmaceutical applications,but none disclosed a dosage form/formulation for the buccal route ofadministration.

We embarked on previously disclosed orally disintegrating tablets (ODTs)of AVA [Broman et al. Orally disintegrating dosage form for theadministration of AVA, and associated methods of manufacture and use.U.S. Pat. No. 10,028,916 B2, Jul. 24, 2018], which was noticed to berapidly dissolved/disintegrated in the oral cavity. It also increasedthe duodenal absorption of AVA. Although ODTs of AVA have a potentialenhancement of bioavailability, they are still administered via the oralroute without avoiding hepatic metabolism. Therefore, we designed AVAbuccal formulations to enhance the bioavailability of AVA and avoid alldrawbacks of the oral route, including the extensive hepatic metabolismfrom the oral route and the consequent repeated daily doses andundesirable side effects. The objective was reflected in enhancing AVAbioavailability and extending the duration over 72 h as displayed by themean plasma concentration-time profiles (FIG. 8 ) after oraladministration of the optimized buccal tablet on healthy humanvolunteers (undisclosed in data revealed from ODTs of AVA). Anotherpatent disclosed an in-situ gel loaded with nanoemulsion forencapsulating any phosphodiesterase type V inhibitors for intramuscularadministration with expected long duration [Mosli et al. In situ gelloaded with phosphodiesterase type V inhibitors nanoemulsion, US2015/0099751 A1, Apr. 9, 2015]. However, this patent undisclosed anyspecific investigations and data regarding AVA encapsulation in thein-situ gel nanoemulsion formulation. The same was found in the patentdisclosed a transdermal formulation for the delivery of sildenafilmainly along with the potential delivery of other phosphodiesterase type5 inhibitors [Fossil et al. Transdermal delivery of sildenafil and otherphosphodiesterase type 5 inhibitors, US 2016/0067252 A1, Mar. 10, 2016]and undisclosed any specific investigations or data regarding AVAloading in the transdermal formulation.

Another invention disclosed transfersome-containing transdermal filmformulations for enhancing AVA efficacy and bioavailability in rats[El-Say et al. Transfersome-containing transdermal film formulations andmethods of use, U.S. Pat. No. 11,185,513 B1, Nov. 30, 2021, A1-hejailiet al. Transdermal film loaded with avanafil ultra-deformablenanovesicles to enhance its percutaneous absorption and bioavailability,AAPS PharmSciTech, 2022 Jan. 4;23(1):46]. This invention showed animproved maximum (peak) plasma concentration and area under the curveover 24 h of the transferosomal-transdermal AVA formulation (254.66ng/mL and 1657.5 ng/mL×h, respectively) compared with the raw AVAtransdermal formulation. In addition, one more novel formulation ofAVA-loaded invasomal transdermal film [Ahmed et al. Development of anoptimized avanafil-loaded invasomal transdermal film. U.S. Pat. No.10,751,294 B1, Aug. 25, 2020, Ahmed et al. Development of an optimizedavanafil-loaded invasomal transdermal film: Ex vivo skin permeation andin vivo evaluation. Int J Pharm. 2019; 570:118657] showed an enhancedmaximum (peak) plasma concentration of AVA in rats and area under thecurve over 24 h (250.39 ng/mL and 1717.04 ng/mL×h, respectively)compared with the raw AVA film. On the other hand, the in-vivo study inhuman volunteers performed on our AVA buccal formulation disclosed amuch higher improvement in AVA bioavailability (FIG. 8 ) with 314.14ng/mL and 11044.78 ng/mL×h of maximum (peak) plasma concentration andarea under the curve over 72 h, respectively.

Moreover, the plasma concentration of AVA versus the time profile of theAVA buccal formulation reflected the extended duration over 72 h,compared with a maximum of 24 h of other AVA formulations investigatedin previous research articles [Alamoudi et al. Investigating thepotential of transdermal delivery of avanafil using vitamin e-tpgs basedmixed micelles loaded films. Pharmaceutics 2021 May 17;13(5):739,Kurakula M, Naveen N. R, Patel B, Manne R, Patel D B. Preparation,optimization, and evaluation of chitosan-based avanafil nanocomplexutilizing antioxidants for enhanced neuroprotective effect on PC12cells. Gels. 2021;7(3), Fahmy et al. Development and evaluation ofavanafil self-nanoemulsifying drug delivery system with rapid onset ofaction and enhanced bioavailability. AAPS PharmSciTech. 2015Feb.;16(1):53-8, Aldawsari et al. Formulation and optimization ofavanafil biodegradable polymeric nanoparticles: A single-dose clinicalpharmacokinetic evaluation. Pharmaceutics. 2020 Jun. 26;12(6):596,Soliman et al. Formulation of avanafil in a solid self-nanoemulsifyingdrug delivery system for enhanced oral delivery, Eur J Pharm Sci. 2016;93:447-55, Gardouh et al. Mixed Avanafil and Dapoxetine Hydrochloridecyclodextrin nano-sponges: Preparation, in-vitro characterization, andbioavailability determination. J Drug Deliv Sci Technol. 2022;68(December 2021):103100, Kurakula et al. Solid lipid nanoparticles fortransdermal delivery of avanafil: optimization, formulation, in-vitroand ex-vivo studies. J Liposome Res. 2016;26(4):288-961. To summarize,this confirmed the superiority of our AVA buccal formulation inenhancing AVA bioavailability with prolonged duration over otherpreviously issued patents and research articles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Solubility of AVA in distilled water after complexation withdifferent PVP polymers (K90, K30, and K18) in different ratios.

FIG. 2 . Powder X-ray diffraction patterns of pure avanafil (top panel),and its solid dispersion with PVP K-90 (1:2 ratio)(bottom panel).

FIG. 3 . In-vitro dissolution profiles of AVA from different buccalformulations.

FIG. 4 . Pareto charts of all independent variables on mucoadhesionstrength, tablet hardness, initial AVA released after 1 h, andcumulative AVA released after 8 h.

FIG. 5 . Estimated three-dimensional response surface plots for theeffect of the studied variables on mucoadhesion strength, tablethardness, initial AVA released after 1 h, and cumulative AVA releasedafter 8 h.

FIG. 6 . FTIR Spectra of pure avanafil (top panel), avanafil soliddispersion with PVP K-90 (2:1 ratio) (middle panel), and the optimizedbuccal tablet (bottom panel).

FIG. 7 . DSC Thermograms of pure avanafil, avanafil solid dispersionwith PVP K-90 (2:1 ratio), and the optimized buccal tablet.

FIG. 8 . The mean plasma concentration-time profiles of AVA after oraladministration of a single dose (50 mg) of the marketed tablet andoptimized buccal tablet, * Significant difference at p<0.05,**Significant difference at p<0.01, **** Significant difference atp<0.0001.

DESCRIPTION OF THE INVENTION

An aspect of the disclosure provides a buccal formulation of avanafilthat demonstrated high bioavailability and prolonged the duration ofaction, reducing the frequency of dosing and minimizing the side effectspotentially beneficial in treating patients with erectile dysfunction.In some embodiments, the solid dispersion of AVA with PVP K90 in theratio of 2:1 showed the highest solubilization capacity of AVA (0.044mg/mL). At the same time, the pure drug solubility in distilled waterwas 0.003 mg/mL.

In another embodiment, converting AVA from crystalline to amorphous formby solid dispersion approach might be the reason behind the highsolubilization and/or subsequent adsorption.

In another embodiment, adding the mucoadhesive polymer to the buccaltablet increases its retention time in the oral cavity and controls thedrug release from the buccal mucosa.

In another embodiment, incorporating sodium deoxycholate as a permeationenhancer increased the permeability through the buccal mucosa and,subsequently systemic delivery.

In another embodiment, the Design of Experiment (DoE) approach inoptimizing the formulation ingredients helped us reach the optimum levelof each component that yields the buccal tablet's optimum qualityattributes.

In another embodiment, the investigation of the optimized buccal AVAtablet on human volunteers rather than laboratory animals confirmed theresults reliability.

In another embodiment, the optimized AVA-buccal tablet improved all thepharmacokinetic parameters compared to the marketed oral tablet.

In other aspects, the optimized buccal AVA tablet significantlyincreased AVA plasma concentrations over the study period, whichindicates the improvement of AVA's relative bioavailability and theprolonged duration of therapeutic action.

Another aspect of the disclosure provides a method of treating malesexual dysfunction in a subject in need thereof, comprisingadministering a formulation as described herein to the subject.

Methodology Preparation of AVA-Solid Dispersions

In this study, the polyvinylpyrrolidone polymer was used to enhance thesolubility of AVA. Different solid dispersion formulations were preparedusing PVP K90, PVP K30, and PVP K18 at three polymer to drug ratios of1:1, 2:1 and 3:1 (w/w). The known weight (50 mg) of AVA was accuratelyweighed and dissolved in 5 mL dimethyl sulfoxide (DMSO) to obtain aclear transparent solution. Similarly, each polymer with the specifiedamount was dissolved in 5 mL methanol separately. The two solutions weremixed and vortexed for 30 min. Subsequently, the mixture was transferredto Büchi-M/HB-140 rotary evaporator (Flawil, St. Gallen, Switzerland)and vacuum dried at 45-50° C. and 22 rpm rotation speed. The dried massfor each AVA-solid dispersion was scrapped, and the obtained AVA-soliddispersion was stored in a desiccator for characterization (33).Solubility study of the elaborated AVA-solid dispersions

A solubility study for the prepared solid dispersions was conductedseparately in distilled water, as described previously, with fulldetails published (34). In screw-capped vials, 10 mL of distilled waterwas placed, and an excess amount of either the pure drug or the preparedsolid dispersions was added. The vials were kept in a shaking waterbath, for 48 h, in a thermostatically controlled shaking water bath(Model 1031; GFL Corporation, Burgwedel, Germany). Samples from the vialwere obtained and were determined in triplicate using spectrophotometricassay at λ_(max) 285 nm, and the data were expressed as the average ±the standard deviation. The AVA-solid dispersion with the highest watersolubility was determined and selected for further investigations.Powder X-ray Diffraction (PXRD)

The crystallinity of the AVA-solid dispersion with the highest watersolubility was investigated using PXRD. Diffractograms for AVA and theselected AVA-solid dispersion were determined using the Ultima IVdiffractometer (Rigaku Inc., Tokyo, Japan) (35). Design of AVA-buccaltablet-formulations

The selected AVA-solid dispersion was incorporated into the design ofthe AVA-buccal tablet formulations. The Box-Behnken design (BBD) wasemployed (36-38) to evaluate the effect of the polymer type as X₁(depending on the polymer charge), the polymer percentage as X₂, and themucopenetration enhancer (Sodium deoxycholate) percentage as X₃ on thequality attributes of the AVA-buccal tablet-formulations. Fifteenexperimental runs were suggested by design. Carbopol as an anionicpolymer, HPMC as a nonionic polymer, or chitosan as a cationic polymerwere studied as X₁. In addition, the level of X₂ was investigated from15 to 25%, while X₃ was studied from 1 to 3% (Table 1).

TABLE 1 Box-Behnken design independent levels and dependent variableswith the specifiedr esponses and assigned goals of AVA-buccaltablet-formulations. Independent variables Levels (Factors) Low MediumHigh Units (A) X₁: Polymer type Carbopol HPMC Chitosan (anionic)(nonionic) (cationic) (B) X₂: Polymer percentage 15 20 25 (%) (C) X₃:Muco-Penetration  1  2  3 (%) enhancer percentage Dependent variables(Responses) Units Goal Y₁: Muco-adhesion strength G Maximize Y₂: Tablethardness N Maximize Y₃: Initial AVA released (%) Maximize after 1 h Y₄:Cumulative AVA (%) Maximize released after 8 h

Statistical analysis was performed using Statgraphics Centurion 18software, Statgraphics Technologies, Inc. (Virginia, Va., USA) toinvestigate the effect of these independent variables on themucoadhesion strength (Y₁), the tablet hardness (Y₂), the initial AVAreleased after 1 h (Y₃) and Cumulative AVA released after 8 h (Y₄). Alack of fit test was also performed to select the best-fitted modelrepresenting responses (39,40). Elaboration of AVA-buccaltablet-formulations

Fifteen AVA-buccal tablets formulations were prepared, as shown in Table2 (see below). Each AVA-buccal tablet-formulation contained a specifiedamount of the selected well-dried AVA-solid dispersion (equivalent to 50mg AVA), a specified amount of the studied polymer (X₂), a predeterminedamount of the mucopenetration enhancer (Sodium deoxycholate; X₃), 10 mgFujsil and 4 mg mannitol. The mixture was mixed well with continuoustrituration for 10 min in a mortar, and the dried mixture was passedthrough 20 mesh sieves. The powder excipients were de-lumpedindividually through a No. 40 mesh sieve. The de-lumped powders weremixed for 15 min. Talc powder and magnesium stearate (0.5%) were alsode-lumped through the 40-mesh sieve and finally added to the powderblend and mixed for 3 min. AVA-buccal tablets were made at 10 KNcompression force in a single punch tablet press (Erweka, GmbH,Heusenstamm, Germany) equipped with 9 mm flat round tooling sets (35).

Characterization of the Elaborated AVA-Buccal Tablet-Formulations

The prepared AVA-buccal tablets were visually inspected for anydrawbacks during the compression and then examined for their qualityattributes, such as weight and content uniformity, thickness,friability, as well as the mucoadhesion strength, tablet hardness, andfor the in-vitro release profile of AVA, according to the requirementsof the United States Pharmacopeia (41). The drug content of theformulated buccal tablets was determined three times usingspectrophotometric assay at λ_(max) 285 nm. Twenty tablets (n=20) fromeach buccal tablet-formulation were weighed using an electronic balance,and their average weight was calculated (initial weight). Then, thetablets were put into the friabilator drum and subjected to 100revolutions. The tablets were taken, freed from dust, and reweighed(final weight). The friability percentage was calculated from Eq. (1).

$\begin{matrix}{{{Friability}(\%)} = {\frac{{Weight}_{initial} - {Weight}_{final}}{{Weight}_{initial}} \times 100}} & (1)\end{matrix}$

Mucoadhesion Strength

Mucoadhesion is a characteristic of a dosage form that can interact withmucosal membranes, especially with their mucin component. Themucoadhesive strength measures the force needed to detach the film fromthe buccal cavity. In this study, the tensile strength method has beenutilized to evaluate the mucoadhesive strength of the prepared buccaltablets (42). This method measures the physical interaction between thefilm and buccal tissue. Results obtained for the mucoadhesive strengthusing the tensile strength apparatus method were used in theexperimental design.

Cow buccal mucosal tissue, obtained from a local slaughter-house, wasused as a model to evaluate the mucoadhesive properties for the preparedtablets using Shimadzu Tensile Strength Machine, EZ-SX withhigh-precision (±0.5%), Shimadzu Co. (Kyoto, Japan). In this experiment,the force required to break the interaction between the prepared tabletand the buccal mucosal tissue was used to assess the mucoadhesivestrength (30). A buccal tissue of 2cm² was fixed on a glass slideattached to the apparatus lower stage (stationary platform). Samplesfrom each buccal tablet of the same surface area were adhered to anotherglass slide using two-sided adhesive tape attached to the apparatusupper platform. The tablet was allowed to interact with the mucosaltissue by applying downward force for 2 minutes before running theexperiment. The crosshead was then raised at a constant speed of 0.5mm/min, and the force required for complete detachment (breakpoint) wasrecorded. Each experiment was repeated three times.

Tablet Hardness

This test investigates a tablet structural integrity and breaking pointand how it alters during storage, transportation, packing, and handlingconditions before use. A hardness tester is used to study thischaracter. Twenty tablets (n=20) from each buccal tablet formulationwere taken and individually placed between the two probes of thehardness tester, one of which is a movable probe and the other is animmovable probe. The force required to break the tablet was recorded,which was taken as the hardness of the tablet (35).

In-Vitro Dissolution Study

A USP dissolution test apparatus type II (paddle type), DT 700 LHdevice, Erweka GmbH DT 700 (Heusen-stamm, Germany) was used to study AVArelease from the prepared tablet formulations. Six tablets (n=6) fromeach formulation were taken and placed individually in a vesselcontaining 900 mL of phosphate buffer of pH 6.8 with 1% sodium laurylsulfate to confirm sink condition (43). The test was performed at 37°C.±0.5° C. at a rate of 50 rpm. Samples were diluted with the buffer,filtered via a 0.45 μm Millipore filter (Millipore Corp., Bedford,Mass., USA), and analyzed spectrophotometrically at 285 nm, using aUV-Vis spectrophotometer (Jenway 7315, Bibby Scientific Limited, Stone,Staffordshire, UK). Samples of 5 mL were withdrawn at specified timeintervals (0.5, 1, 2, 3, 4, 6, and 8 h), and the same volume wasreplaced with a fresh medium. Prediction of the optimized AVA-buccaltablet-formulation

Analysis of variance and multiple response optimization were utilizedfor predicting the optimized AVA-buccal tablet using the statisticalpackage Statgraphics® Centurion 18 Software (StatPoint, Inc., Warrenton,Va., USA). Characterization of the optimized AVA-buccaltablet-formulation

The optimized formulation was prepared and evaluated for themucoadhesion strength, tablet hardness, and the in-vitro dissolutionstudy (release profile of AVA from the optimized AVA-buccal tablet).This optimized formulation was also characterized via X-ray Diffraction,Fourier Transform Infrared Spectroscopy, and Differential ScanningCalorimetry then scaled up to be evaluated in-vivo for itspharmacokinetic parameters on human volunteers. Fourier TransformInfrared Spectroscopy (FT-IR)

To investigate any potential interaction between AVA and the studiedpolymer used to develop the solid dispersion as well as the tablet'sexcipients in the optimized-buccal tablet-formulation, FT-IR spectrawere recorded using a Nicolet iS10, Thermo Scientific Inc. (Waltham,Mass., USA) (44).

Differential Scanning Calorimetry (DSC)

DSC was conducted to evaluate AVA's thermotropic characteristics andthermal performance, the selected AVA-solid dispersion, and theoptimized AVA-buccal tablet-formulation using a DSC 8000, PerkinElmer,Inc. (Waltham, Mass., USA). About 5 mg of the sample was sealed inaluminum pans and heated at the rate of 10° C./min in a temperaturerange of 25-400° C. under a nitrogen atmosphere at a flow rate of 100mL/min (45).

In-Vivo Pharmacokinetic Evaluation on Healthy Human Volunteers

In this work, an oral pharmacokinetic study was carried out for theoptimized AVA-buccal tablet-formulation (test) compared with themarketed tablet (reference) on healthy human volunteers.

Study Design and Conduct

An open-label, single dose, randomized, one-period, parallel design, andone-treatment under fasting conditions comprising fourteen days ofscreening preceding 24 h study periods were used. The participants wereadministered a buccal 50 mg dose of AVA from the optimized formulationtablet (test). At the same time, the marketed tablets (reference) wereadministered the same dose orally with water. The study was carried outat the International Center for Bioavailability, Pharmaceutical, andClinical Research. (ICBR), Cairo, Egypt. The Institutional ReviewBoard/Independent Ethics Committee (IRB/IEC) at ICBR formally reviewedthe objective, design, conduct, and analysis for the proposed study andapproved the study protocol on Jul. 18, 2019, with the Ethical ApprovalCode (RESH-007). The study was accomplished in agreement with theDeclaration of Helsinki and the International Conference onHarmonization of Good Clinical Practices. The study was performedfollowing European Medicines Agency (EMA), International Conference onHarmonization (ICH), Good Clinical Practice (GCP), and Food and DrugAdministration (FDA) guidelines. Six subjects per group were assignedand gave their written informed consent before participation in thisstudy. The selected subjects were in good health as determined by theircomprehensive medical histories, conducting physical examinations, vitalsigns, and complete laboratory investigations (hematology, biochemistry,and urine analysis). They were also screened for viral infections andremained under close medical supervision before, during, and after thestudy period. Each subject fasted for at least 12 h before administeringthe studied tablets. Subjects were kept in-house for 72 h before andafter administration of the drug so that regular blood sampling could bewithdrawn at a predetermined time (as described in the Blood Samplingsection).

Subjects

Twelve healthy Egyptian male volunteers participated in the study. Thesubjects' age and body mass index (BMI) ranged from 21 to 30 years and20 to 30 kg/m², respectively. Their median height was 172±5.3 cm.Subjects were classified into two groups (6 per group); the first groupwas administered the optimized AVA-buccal tablet, and the second groupwas given the oral commercial tablets, namely; Stendra®, MetuchenPharmaceuticals, LLC (Freehold, N.J., USA).

Blood Sampling

5 mL blood samples were taken and collected in heparinized tubes beforeand 0.08, 0.17, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 4, 6, 8, 10, 12,24, 48, and 72 h following oral administration of the test and referencetablets. The collected samples were centrifuged at 3000 rpm for 5 min,and the plasma samples were collected and stored at -20°C. untilanalysis.

Chromatographic Conditions

A high-performance liquid chromatography (HPLC) method was developed atEgyptian Research and Development Company laboratories to analyze AVA inhuman plasma. The method was validated according to the FDABio-analytical Method Validation Guidelines 2003 (29). The linearity ofthe method was studied within the concentration range of 10-600 ng/mL,with a regression coefficient (R²)=0.998. Azithromycin was used as aninternal standard. The obtained results for method validation werewithin the acceptance criteria as indicated in the recommendedguidelines. The described method proved to be sensitive, accurate andreproducible, with a lower limit of AVA quantification of 10 ng/mL. TheHPLC-MS/MS-system consisted of Agilent series 1200 of AgilentTechnologies Inc. (Santa Clara, Calif., USA). The system is equippedwith a quaternary pump (G1311A), an autosampler (G1329A), a vacuumdegasser (G1322A), and an ESI electrospray ionization ion source. MassHunter software was used. The mobile phase consisted of acetonitrile 50%and ammonium formate 10 mmole 50%. The flow rate was adjusted at 1mL/min A reverse phase Intersil ODS-3 (4.6 mm×50 cm, dp 5 μm) column ofGL Sciences (Tokyo, Japan) was used at 25° C.

Pharmacokinetic Data Analysis

A non-compartmental pharmacokinetic model using PKsolver (An add-inprogram for pharmacokinetic data) was used to calculate thepharmacokinetic parameters of AVA. Maximum (peak) plasma concentrationover the time specified (C_(max)), time point to reach the maximumplasma concentration (T_(max)), and the area under the plasmaconcentration-time curve from zero time to the last measurableconcentration (AUC_(0-t)) were calculated by the linear trapezoidalmethod. In addition, individual estimates were made of the terminalelimination rate constant (K_(el)), the mean residence time (MRT_(0-∞)),which was calculated by the ratio of AUMC to AUC, and the eliminationhalf-life (t½ _(/2)), which was calculated as 0.693/K_(el). Moreover,the apparent total body clearance of the drug after oral administration(Cl) was calculated by dividing the dose by AUC, and the apparent volumeof distribution during the terminal phase after non-intravenousadministration (V_(d)) was calculated by multiplying total bodyclearance by MRT. Finally, the relative bioavailability of the optimizedAVA-buccal tablet (AUC_(test)/AUC_(reference)×100) was determined (46).

Statistical Analysis

GraphPad Prism, version 8.4.2 Software (San Diego, Calif., USA) was usedto analyze all the obtained data statistically. The solubility studyanalysis was made using a one-way ANOVA/Tukey-Kramer post-hoc test atP<0.05, and data are expressed as mean ±SD. Regarding the plasmaconcentration-time curve, two-way ANOVA followed by Sidak's multiplecomparisons test was conducted to compare each mean with the others ateach time point and assess the significance between groups. At the sametime, a two-tailed unpaired t-test was used to assess thepharmacokinetic parameters of the formulations. Results with P<0.05 wereconsidered significant.

RESULTS AND DISCUSSION Solubility study of the Elaborated AVA-SolidDispersions

The selection of PVP polymers for enhancing AVA solubility was based onthe previously confirmed reports for the superiority of these polymers,which particularly improved AVA solubility (36). FIG. 1 illustrates thesolubility of AVA in distilled water after complexation with differentPVP polymers (K90, K30, and K18) in different ratios. PVP K90: AVA withthe ratio 2:1 showed the highest solubilization capacity of AVA (0.044mg/mL), while the pure drug solubility in distilled water was 0.003mg/mL, which confirms that AVA is practically insoluble in wateraccording to USP, which describes the substance that needs more than10,000 mL to dissolve 1 g with the practically insoluble one. It is alsonoted that PVP K90: AVA-solid dispersion (2:1) significantly increasedAVA solubility with a 0.0033 P-value over PVP K18: AVA-solid dispersion(3:1) and with P-value<0.0001 over all other ratios and other types ofused polymers as well (FIG. 1 ).

Powder X-ray Diffraction (PXRD)

PXRD was used to determine the crystalline state of AVA in its purestate and PVP K90: AVA (2:1)-solid dispersion. It measures thedisappearance of constructive, specific peaks of drugs in the soliddispersion and retaining peaks of the polymer material. The PXRD patternin FIG. 2 of the pure drug (AVA) shows sharp diffraction peaks at 7.3,13.6, 17.9, 19.3, and 21.3° with high intensity, which indicates thatthe pure drug was present in the crystalline state. The decrease in thenumber and intensity of the characteristic peaks in the XRD pattern ofthe solid dispersion, as illustrated in FIG. 2 , indicates theconversion of AVA from crystalline to amorphous form. This lack ofcrystallinity in the formulation might be due to AVA solubilizationand/or subsequent adsorption on the studied PVP K90 polymer (47,48). Thesolubilization or amorphization of the drug in the solid dispersionleads to the resulting improvement in the apparent solubility that hasbeen confirmed in the previous section.

Consequently, PVP K90: AVA (2:1)-solid dispersion has been selected forfurther inclusion in the buccal tablet formulation design.

Formulation and Evaluation of the AVA-Buccal Tablet-Formulations

Fifteen AVA-buccal tablet-formulations were prepared as suggested by BBD(Table 2). Quality control tests of the prepared AVA-buccaltablet-formulations revealed that the AVA content of all formulationswas found to be in the range of 85.58% to 113.26% in F14 and F1,respectively. These results were compiled with USP's officialspecifications and reflected the weight uniformity in all formulations(41). In addition, there is no observed variation in the thickness ofall formulations. The friability and the hardness of all tabletformulations ranged from 0.01-0.062%, and 96.67-167 N, respectively,which complied with BP friability test limits (<1%). The friability andhardness results reflected the prepared buccal tablets' acceptablemechanical properties and good breaking strength. Moreover, themucoadhesion strength of the prepared AVA-buccal tablet-formulations wasfound to be from 129.21 to 351.85 G in F-6 and F-4, respectively. Fromthe in-vitro dissolution study (FIG. 3 ), the percentages of initialrelease were varied from 4.843% for F12 to 12.173% for F1, while thepercentages of cumulative release were ranged from 83.005% for F12 to110.71% for F1. In the following sections, full analysis andelucidations regarding the selected quality attributes and the optimizedformulation's prediction will be discussed in detail and thoroughly.Response surface methodology for optimization of the formulations

RSM has been widely used in the formulation development of modernproducts and for modifying existing products. It produces polynomialequations and maps the responses over formulation variables to determinethe optimum formulation (49). This study utilized the RSM to recognizethe influence of the dependent variables (X₁, X₂, and X₃) on the studiedresponse variables (Y₁, Y₂, Y₃, and Y₄). Table 2 lists the BBD matrixthat involves all suggested formulations' independent and dependentvariables.

TABLE 2 Composition of Box-Behnken design for AVA-buccaltablet-formulations and observed values of the studied responses.Factors Responses ** X₂ X₃ Y₁ Y₂ Y₃ Y₄ Formulations* X₁ (%) (%) G N (%)(%) F1 HPMC 15.0 3.0 207.54 116.33 12.173 110.71 F2 HPMC 25.0 1.0 152.24160 9.207 95.809 F3 HPMC 25.0 3.0 333.23 167 10.108 101.492 F4 Carbopol15.0 2.0 351.85 114 9.684 98.737 F5 Chitosan 20.0 1.0 202.45 125.3310.001 99.959 F6 Carbopol 20.0 1.0 129.21 103 5.068 85.906 F7 Chitosan25.0 2.0 271.66 134 7.119 86.183 F8 Chitosan 20.0 3.0 217.84 127.338.873 96.204 F9 Chitosan 15.0 2.0 323.14 106.33 7.552 96.716 F10Carbopol 20.0 3.0 254.74 117.33 9.932 99.753 F11 HPMC 15.0 1.0 316.5196.67 8.086 98.804 F12 Carbopol 25.0 2.0 256.58 136.33 4.843 83.005 F13HPMC 20.0 2.0 245.82 146 7.212 92.149 F14 HPMC 20.0 2.0 286.54 141 6.52394.369 F15 HPMC 20.0 2.0 247.50 148.33 7.362 93.661 *Each 200 mgAVA-buccal tablet-formulation contains 150 mg PVP-K90: AVA-soliddispersion (2:1), specified amount of selected polymer (X₂), specifiedamount of muco-penetration enhancer (Sodium deoxycholate; X₃), 10 mgFujsil and 4 mg Mannitol. **Values of responses are the means oftriplicate measurements for each response and SD values did not exceed5% of the stated values.

The mucoadhesion strength, tablet hardness, and the in-vitro dissolutionprofile (the selected quality attributes as dependentvariables/responses) were investigated using a 3-factor, 3-level BBDmodel. This model was employed to inspect, augment, and evaluate thequadratic and interaction effects of the selected factors on theresponses. In addition, polynomial equations and a lack of fit testsummary data were generated and discussed in the following sections.

Lack of Fit Test

The lack of fit test is premeditated to reveal the suitability of theselected model to demonstrate and represent the detected data. In thecase of the P-value for lack of fit in the ANOVA results that is morethan or equal to 0.05, the model seems suitable for the detected data atthe 95.0% confidence level (50,51). The lack of fit test summary datathat is displayed in Table 3 revealed that the selected model for allresponses has an insignificant lack of fit (all P-values are more than0.05).

TABLE 3 Parameters of lack-of-fit tests of responses. Lack-of-fitparameters Sum of Degree of Mean F- Lack of Fit Responses SquaresFreedom Square ratio p-value Y₁ 2368.01 3 789.338  1.49 0.4263 Y₂749.317 3 249.772 17.81 0.0536 Y₃ 4.27866 3 1.42622  7.12 0.1256 Y₄42.6288 3 14.2096 11.05 0.0841Consequently, and according to these findings, the Quadratic modelselected to represent the observed data can be considered a suitablemodel for all responses utilized in the design of experiment conductedin this research work.

Quantitative Estimation of Factors' Effect

The relevancy-oriented mathematical treatment among the selected factorsand the detected responses were expressed as polynomial equations andexplained for their significance by ANOVA. The estimated factors'effects and the corresponding P-values obtained from ANOVA for all thestudied responses are represented in Table 4.

TABLE 4 Estimated effects of factors and associated P-values for theresponses of AVA-buccal tablet-formulations. Factors X₁ X₂ X₃ X₁ ² X₁X₂X₁X₃ X₂ ² X₂X₃ X₃ ² Y₁ Factor 5.68 −46.33 53.237 −10.476 21.896 −55.07192.175 144.98 −107.321 effect p-values 0.76 0.1046 0.0823 0.7049 0.44230.1394 0.0615 0.0243* 0.0465* Y₂ Factor 5.583 41 10.748 −39.198 2.67−6.165 −5.693 −6.33 −14.528 effect p-values 0.1696 0.0041* 0.05570.0097* 0.5499 0.2415 0.282 0.2331 0.0651 Y₃ Factor 1.0045 −1.5545 2.181−1.15783 2.204 −2.996 1.69217 −1.593 4.03017 effect p-values 0.08650.039* 0.0204* 0.1308 0.0388* 0.0216* 0.0681 0.0706 0.0131* Y₄ Factor2.91525 −9.6195 6.92025 −8.481 2.5995 −8.801 4.0155 −3.1115 12.606effect p-values 0.068 0.0069* 0.0132* 0.0188* 0.1489 0.0162* 0.07660.1111 0.0087* X₁ is the Polymer type, X₂ is the Polymer percentage (%),X₃ is the Muco-Penetration enhancer percentage (%), X₁X₂, X₁X₃, X₂X₃ arethe interaction terms between the factors, X₁ ², X₂ ², X₂ ³ are thequadratic terms of the factors, Y₁ is the Muco-adhesion strength (G), Y₂is the Tablet hardness (N), Y₃ is the Initial AVA released after 1 h(%), and Y₄ is the cumulative AVA released after 8 h (%). *Significanteffect of factors on individual responses.The effectiveness of any factor will be considered significant in casethe effect value is more than zero and its P-value is not equal to ormore than 0.05. Besides, the synergistic factor effect will be labeledwith a positive sign, whereas a negative sign symbolizes theantagonistic effect.

According to the given outcomes, the percentage of the polymer used todevelop the buccal tablets formulation (X₂) was found to significantlyinfluence the tablet hardness (Y₂) synergistically at a P-value of0.0041. X₂ was also found to significantly and antagonistically affectthe initial and cumulative amount of AVA released after 1 h (Y₃) and 8 h(Y₄) at P-values of 0.039 and 0.0069, respectively. The percentage ofmucopenetration enhancer (X₃) was significantly influenced by Y₃(P-value of 0.0204) and Y₄ (P-value of 0.0132). Although the type/chargeof polymer (X₁) was found to exhibit a non-significant effect on allresponses, it was noted that the quadratic effect of X₁ (X₁ ²)negatively influenced both Y₂ and Y₄ with P-values of 0.0097 and 0.0188,respectively. Furthermore, Y₃ was influenced synergistically (P=0.0388)by the interaction term (X₁X₂), while the interaction term (X₁X₃) had anantagonistic effect on both Y₃ and Y₄ (0.0216 and 0.0162 P-values,respectively). In addition, it was noted that the quadratic term of X₃(X₃ ²) had a noticeable negative effect on the mucoadhesion strength(Y1) with a P-value of 0.0465, and a positive effect on Y₃ and Y₄ atP-values of 0.0131 and 0.0087, respectively. In addition, a significantagonistic effect on Y₁ (P=0.0243) was exhibited by the interaction term(X₂X₃). The quadratic term of X₂ (X₂ ²) was found to have no significanteffects on any of the studied responses (Table 4).

Effect of the Independent Variables on Mucoadhesion Strength (Y₁)

The mucoadhesion strength test was performed to measure the ability ofthe prepared buccal tablets to interact with the buccal epithelialcells, which in turn plays an important role in the formulationabsorptivity and bioavailability. Carbopol, chitosan, and HPMC have beenverified for their efficacy as mucoadhesive polymers in pharmaceuticalresearch (52-54).

From the data presented in Table 4, the mucoadhesion strength (Y₁) wasfound to range from 129.21-351.85 G in F-6 and F-4, respectively. Y₁ waspositively influenced by the interaction term of X₂ and X₃ (X₂X₃) andnegatively influenced by the quadratic term of X₃ (X₃ ²). The Paretochart and contour response surface plot of Y₁ (FIGS. 4 and 5 ) alsoconfirmed this finding. As demonstrated in F1 and F3, at fixed levels ofX₁ and X₃ (HPMC buccal tablets with 3% sodium deoxycholate; high levelof mucopenetration enhancer), the increase in X₂ from 15 to 25% wasaccompanied by an increase in Y₁ from 207.54 to 333.23 G. Also, theincrease in X₃ from 1 to 3% in F6 and F10 resulted in an increase in Y₁from 129.21 to 254.74 G at constant levels of X₁ and X₂ (20% Carbopolbuccal tablets). To better describe this complex interaction, theincrease in X₂ at high levels of X₃ will result in an increase in Y₁.Also, the increase in X₃ at mid-high levels of X₂ will increase Y₁. Theinteraction between the polymer used and sodium deoxycholate, used as amucopenetration enhancer, was found to depend on different propertiessuch as; the charge of the polymer, the number of sites available forbinding with sodium deoxycholate, and the hydrophilic/hydrophobiccharacteristics of the studied polymer (55,56). This complexation iswell-known and directly related to the intricate mutual effect of X₂ andX₃ on the mucoadhesion property of tablets.

Effect of the Independent Variables on the Tablet Hardness (Y₂)

Hardness is a crucial test that is used to evaluate the mechanicaldurability of formulated tablets. Table 2 shows the results of thehardness for the prepared buccal tablets. The results ranged from 96.67to 167 N for F11 and F3, respectively.

The estimated effects of factors and the associated p-values aredisplayed in Table 4, while FIGS. 4 and 5 demonstrate the Pareto chartand contour response surface plot of Y₂. ANOVA results exposed that thetablet hardness (Y₂) was highly influenced by the polymer percentage(X₂) with a p-value of 0.0041. As the X₂ was increased from 15 to 25% atthe same level as the other factors, the hardness was increased from96.67 to 160 N in F11 and F2, respectively, and from 106.33 to 134 n inF9 and F7, respectively, which an indication of the synergistic effectof X₂ on Y₂. This trend was also confirmed by the decrease in thehardness from 136.33 to 114 in F12 and F4, respectively, as X₂ decreasedfrom 25 to 15%. This effect is expected since all the studied polymersthat have been utilized in buccal tablets' formulations have been usedas binders in tablet manufacturing (57-60). Therefore, increasing theconcentration of these polymers will lead to an increase in tablethardness. On the other hand, the quadratic term of X₁ (X₁ ²) also had anegative influence on Y₂.

Influences of the Independent Variables on AVA Release (Y₃ and Y₄)

In this study, the initial percentage of drug release after 1 h (Y₃) andthe cumulative percentage of drug release after 8 h (Y₄) were studiedafter investigation of the drug in vitro release profile displayed inTable 2 and FIG. 3 . The highest initial and cumulative AVA released wasobtained from the HPMC-based buccal tablet namely; F1 (12.173% and110.71%, respectively), while the lowest initial and cumulative AVAreleased was observed from the Carbopol-based buccal tablet namely; F12(4.843% and 83.005%, respectively). It was also noted that F3“HPMC-based buccal tablet” ranked second in the highest initial andcumulative AVA release (10.108% and 101.492%, respectively). The secondlowest initial and cumulative AVA released was obtained from F6“Carbopol-based buccal tablet” (5.068% and 85.906%, respectively). Ingeneral, HPMC-based buccal tablets illustrated superiority in their drugrelease compared to the other polymer-based buccal tablets. Datarepresented in Table 4 and the graphical illustration of the Paretochart and contour response surface plot for Y₃ and Y₄ (FIGS. 4 and 5 )revealed that the type of polymer (X₁) did not have a significant effecton the release profile of AVA. In addition, the polymer percentage (X₂)was found to have a significant antagonistic effect on Y₃ and Y₄.According to the data obtained for F9 and F7, the increase in polymerpercentage from 15 to 25% was accompanied by a decrease in the initialrelease percentage from 7.552% to 7.119%, respectively. The cumulativedrug release percentage decreased from 96.716% to 86.183%, respectively,at the same levels of X₁ and X₃. Furthermore, it was also noticed thatthe percentage of mucopenetration enhancer (X₃) had a significantsynergistic effect on both Y₃ and Y₄. Both F1 and F3 were elaboratedwith 3% sodium deoxycholate (highest X₃ value), which is reasonable toexhibit the maximum release profiles in such formulations. On the otherhand, carbopol-based buccal tablets demonstrated the lowest drug releaseprofile, the effect that may be attributed to the carbopol-PVPinterpolymer complexation that potentially resulted in achieving slowerdrug release (61), especially with the comparatively mid-level of themucopenetration enhancer percentage. Moreover, the antagonistic effectof the interaction term (X₁X₃) and the synergistic effect of thequadratic term of X₃ (X₃ ²) were also found to significantly influenceY₃ and Y₄.

Statistical Analysis and Mathematical Modeling of the Experimental Data

After investigating and analyzing the influences of the studiedindependent variables on the selected responses, mathematical modelingfor each response was generated. The best-fit method developed thefollowing equations (Eqs. 2-5) that describe the analysis outcomes ofthe multiple linear regression. Mucoadhesion strength (Y₁)=1402.06+14.12 X₁−107.37 X₂−48.7 X₃−5.24 X₁ ²+2.19 X₁X₂−27.54 X₁X₃+1.84 X₂ ²+14.498 X₂X₃−53.66 X₃ ²   (2)Tablet hardness (Y₂)=−47.55+3.62 X₁+9.92 X₂+47.09 X₃ −19.599 X₁ ²+0.267 X₁X₂−3.08 X₁X₃−0.114 X₂ ²−0.633 X₂X₃−7.264 X₃ ²   (3)Initial AVA released after 1 h (Y₃)=23.186−0.91 X₁−1.19 X₂−3.78 X₃−0.579 X₁ ²+0.22 X₁X₂−1.498 X₁X₃+0.034 X₂ ²−0.159 X₂X₃+2.015 X₃ ²   (4)Cumulative AVA released after 8 h (Y₄)=150.602+5.06 X₁3.55 X₂−15.53 X₃−4.24 X₁ ²+0.26 X₁X₂−4.4 X₁X₃+0.08 X₂ ²−0.31 X₂X₃+6.3 X₃ ²   (5)

Elaboration and Characterization of the Optimized AVA-BuccalTablet-Formulation

Multiple response optimization produces an optimized AVA-buccaltablet-formulation that achieves the study goal. The optimizedparameters were anticipated and investigated to compromise amongstresponses to get a combination of factors' levels that intensify thedesirability function over the study goals (62). The optimized levels ofindependent factors were identified, and the optimum formulation wasfound to have HPMC as the optimum polymer type, 25% and 3% of X₁, X₂,and X₃, respectively. The optimized AVA-buccal tablet-formulation with0.726 optimum desirability value was prepared and assessed as previouslyillustrated in the earlier section. The observed values for Y₁, Y₂, Y₃,and Y₄ were 347.33 G, 166.8 N, 11.98%, and 99.67%, respectively, whilethe predicted values of these responses were 329.5 G, 157.463 N, 9.396%,and 98.911%, respectively.

Fourier-Transform Infrared Spectroscopy

FIG. 6 shows the FTIR spectra of raw AVA powder, PVP K90: AVA(2:1)-solid dispersion, and the optimized AVA-buccal tablet-formulation.Absorption peaks properties for AVA were recorded in the 1700-450 cm⁻¹range. This spectral range contains 1656 to 740 cm⁻¹ domains, which isimportant for finding AVA analog. A specific AVA absorption band wasobserved at 1657 cm⁻¹, which is attributed to the carbonyl group of theamide bond (HN-C=O). Another drug peak was detected at 1435 cm^(-1,)related to the stretching vibration of the C-N and a band at 743 cm⁻¹represent the benzene (63). It is also observed from FIG. 6 that theabsorption bands of both PVP K90: AVA (2:1)-solid dispersion and theoptimized AVA-buccal tablet-formulation did not show interference withthe characteristic drug peaks, indicating the absence of chemicalinteraction between the drug and the components used to develop bothformulations (solid dispersion and the studied tablet). The followingsection will explain more explanation for the interaction between AVAand the studied components in the two formulations (DSC).

Differential Scanning Calorimetry

FIG. 7 shows the DSC thermograms of raw AVA powder, PVP K90: AVA(2:1)-solid dispersion, and the optimized AVA-buccal tablet-formulation.The thermogram of AVA showed a sharp endothermic peak at 162° C.corresponding to the melting point of AVA. This peak indicates thecrystalline nature of AVA. However, the characteristic peak of AVAdisappeared in the DSC thermograms of the PVP K90: AVA (2:1)-soliddispersion and the optimized AVA-buccal tablet-formulation (FIG. 7 ),which is an indication of the possible drug physical change upon mixingwith the solid dispersion ingredient (PVP K90) (64) and the buccaltablet excipients (48,65-68). This behavior may be attributed to thecomplete solubilization of the drug in the form of an amorphous state inthe solid dispersion and buccal tablet mixture, as previously mentioned(69). The absence of the endothermic peak can also be attributed to thesuppression of the thermal feature of the drug because of the formationof an amorphous solid solution (35).

In-Vivo Pharmacokinetic Evaluation on Healthy Human Volunteers

All volunteers who participated in the study have fully completed theclinical research. The pharmacokinetic parameters of the clinical studyare depicted in Table 5.

TABLE 5 In-vivo pharmacokinetic parameters of the optimized AVA buccaltablet and commercial AVA oral tablet. Parameters (unit) Optimized AVAbuccal tablet (±SD) Commercial AVA oral tablet (±SD) K_(el) (h⁻¹) 0.035(±0.009) 0.174 (±0.112) t_(1/2) (h) 20.528 (±5.514) 7.049 (±7.181)T_(max) (h) 8.667 ** (±2.309) 0.667 (±0.289) C_(max) (ng/mL) 314.137(±19.03)   295 (±65.383) AUC_(0-t) (ng/mL × h) 11044.778 **** (±892)1026.213 (±41.155)   AUC_(0-∞) (ng/mL × h) 12475.987 *** (±1362.53)1138.002 (±143.119)  AUMC_(0-∞) (ng/mL × h²) 437840.2 ** (±117249.207)8485.468 (±7528.117) MRT_(0-∞) (h) 34.77 ** (±6.401) 7.012 (±5.388)V_(d) [(mg/kg)/(ng/mL)] 0.118 (±0.023) 0.416 (±0.378) Cl([mg/kg)/(ng/mL)/h) 0.004 *** (±4.705 × 10⁻⁴) 0.044 (±0.005) Note: *,**, ***, and **** denote Significant different of values of theoptimized AVA buccal tablet versus values of the oral commercial AVAtablet at P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

FIG. 8 displayed the plasma concentration-time curve profiles after oraladministration of the optimized AVA-buccal tablet compared to themarketed oral tablets. The results indicated that AVA's maximum plasmaconcentration (C_(max)) in the optimized AVA-buccal tablet was 314.137ng/mL. This C_(max) was obtained within 8.67 h (T_(max)).

On the other hand, the commercial oral tablets demonstrated a C_(max) of295 ng/mL after 0.67 h. Although the C_(max) of AVA in the optimizedAVA-buccal tablet was not significantly different from that of thecommercial oral tablet, the optimized AVA-buccal tablet reached themaximum plasma concentration 8 h after the commercial oral tablet. Inaddition, the optimized AVA-buccal tablet produced significantly higherAVA plasma concentrations than the commercial oral tablet 4 h afteradministration to 48 h, confirming the prolonged therapeutic actionduration. Amazingly, the optimized AVA-buccal tablet showed higher AUCcompared to the commercial oral tablets, which indicates the improvementof the relative bioavailability of AVA in the optimized buccal tablet by1076.27% over the commercial oral tablet. Furthermore, the two-tailedunpaired t-test revealed that MRT_(0-∞) of the optimized AVA-buccaltablet was found to be 4.96 folds higher than that of the commercialoral tablet (P<0.01), while the clearance (Cl) of the optimizedAVA-buccal tablet was found to be 11 times lower than the Cl of thecommercial oral tablet (P<0.001).

The improved absorption, bioavailability, and prolonged extent of theoptimized AVA-buccal tablet can be understood through two correlatedapproaches. The first approach is the enhanced solubilization of thedrug via utilization of PVP K90 solid dispersion, as well as the use ofsodium deoxycholate as mucopenetration enhancer (70), and thus moreavailability of AVA to be absorbed, which is considered the firstrate-limiting step for drug absorption in the BCS Class II compounds(71). The second approach is using the buccal route for delivery,depending on the limited surface area of the buccal cavity forabsorption in comparison to the oral route (15). This feature isintentionally utilized to control the absorption and decrease theincidence of prompt high AVA plasma concentrations. In addition toutilizing this feature, using modified/optimized release mucoadhesivebuccal tablets can be considered the second rate-limiting step for drugabsorption (72). Therefore, changing the drug release from the optimizedmucoadhesive AVA-buccal tablet to release the drug load over 8 hsucceeded in achieving the revealed extended absorption of AVA over 72h. This finding is expected to maximize the benefits, especially aftercircumventing the hepatic metabolism via the buccal route ofadministration.

Despite the fast onset achieved by the commercial oral tablet, it cannotbe considered a superior effect over the 8 h delay in T_(max) for theoptimized AVA-buccal tablet. The latter behavior can potentially confirmmuch better control for AVA's therapeutic/pharmacological activity overa longer period. According to the chronic nature of any erectiledysfunction disorder (which requires treatment for 3 months up to 1-yearduration (73,74)), the enhanced bioavailability via this novel approach,along with the prolonged duration of efficacy, will improve theconvenience, adherence, and compliance for patients with erectiledysfunction.

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Acknowledgment of Sponsored Research

This invention was funded by the Deanship of Scientific Research (DSR)at King Abdulaziz University, Jeddah, under grant no. (RG-39-166-43).Therefore, inventors acknowledge with thanks the DSR for technical andfinancial support.

The invention claimed is:
 1. A buccal tablet formulation, comprising 100mg of polyvinylpyrrolidone K-90 (PVP K-90); 36 mg of hydroxypropylmethylcellulose functioning in said formulation as a mucoadhesivepolymer; a sodium deoxycholate functioning in said formulation as amucopenetration enhancer; 10 mg of porous silicon dioxide; 4 mg ofmannitol; and 50 mg of avanafil, wherein the PVP K-90 to avanafil ratioranges from 2.3:1 to 1.7:1.
 2. The buccal tablet formulation of claim 1wherein the PVP K-90 to avanafil ratio is approximately 2:1.
 3. A methodof treating erectile dysfunction in a subject in need thereof,comprising administering a therapeutically effective amount of theformulation of claim 1 to the subject.